Exciting progress of cancer immunotherapy focusing on immune checkpoints

. Immune checkpoints blockade (ICB) has made revolutionary progress in cancer therapy recently. The development of blocking agents to checkpoints on coinhibitory pathway, which prevents inflammation-induced tissue damage but also induces the cancer immune evasion, and retrieves the productive immune responses against tumors. The striking clinical trial results of ICB, by targeting the cytotoxic T lymphocyte– associated protein 4 (CTLA-4), the programmed cell death 1 (PD-1) or PD-1 ligand 1 (PD-L1), has promoted the approval of multiple antibodies for several cancer types by the US Food and Drug Administration (FDA). In addition, the combination of multiple types of blockade even increased the efficacy of tumor regression. Following the previous success, other immune checkpoints have also been verified, such as lymphocyte-activated gene-3 (LAG-3) and Signal-regulatory Protein alpha (SIRPα). However, not all patients can get benefits from ICB and the mechanisms of these coinhibitory pathways are not quite clear. Therefore, understanding the mechanisms of ICB is a formidable challenge that could have far reaching guidelines for therapeutic strategies in cancer. This article summarizes the literature to date regarding ICB that may help more patients get benefit from immunotherapy.


INTRODUCTION
Immune checkpoints like CTLA-4, PD-1, LAG-3, and SIRP-α are discovered to be used by tumor cells as tools to suppress the immune system. Monoclonal antibodies were developed to block the signaling between those molecules and their ligands. Most of those Abs showed significant effect on the regression of the tumor cells on clinical trials and some of them were officially proved and created as commercials. In this article, we are going to introduce some of the immune checkpoints and their mechanisms, and present their functions as therapeutic targets.

CTLA-4
CTLA-4 (CD152) was discovered as a negative regulator for T-lymphocyte in 1985 and later identified in 1987. CTLA-4 is a homolog of T-lymphocytes co-stimulatory receptor CD28, and they bind to the same ligands B7-1(CD80) and B7-2(CD86), but CTLA-4 has a higher affinity to those ligands. [1] CTLA-4 inhibits T cell response in an intrinsic and an extrinsic manner. (1) In a T cell, CTLA-4 can intrinsically outcompete with CD28 for B7 ligand binding or it recruits phosphatases to the cytoplasmic area of CTLA-4, which results in a reduced TCR and CD28 signaling. [2] (2) CTLA-4 on T regulatory cells can extrinsically inhibit the activation of other T cells by reducing the expression of B7-1 and B7-2 on Antigen Presenting Cells. T regulatory cells can release cytokines like IL-10 causing the downregulation of B7 [3] or remove them from APCs through trans-endocytosis, which results in a reduced interaction between CD28 and B7. [4] Numerous studies have shown that anti-CTLA-4 blocking antibodies can facilitate tumor regression, long term immunity, and antitumor response. The anti-CTLA-4 blocking antibodies target both effector T cells and regulatory T cells; CD8+ T cells are always required for therapeutic benefit, but CD4+ T cells are only needed in some mouse models. [4] Recent works have shown two effects of anti-CTLA-4 blocking antibodies. They block the interaction of CTLA-4 pathways and decrease the number of regulatory T cells in the tumor microenvironment. [4,5] The ratio of CD8+ TILs/Tregs has a positive correlation with overall survival of patients. [6] In a ovarian cancer microenvironment, CTLA-4 blocking antibody promoted the TILs growth in majority of patients (12/14) compared to 64% of patients when tumor fragments were cultured in IL-2 standard condition. [7] The two anti-CTLA-4 monoclonal antibodies ipilimumab and tremelimumab show great effects in immunotherapy and establish more ways to promote the depletion of tumors. Patients who received the combination of gp100 peptide vaccine and ipilimumab shows a higher survival advantage than only receiving the treating of gp100 peptide. [4,8] A second phase 3 trial, the combination of dacarbazine and ipilimumab compared to dacarbazine alone shows an overall greater survival rate at a 3 years to 1 year on E3S Web of Conferences 185, 03007 (2020) ICEEB 2020 http://doi.org/10.1051/e3sconf/202018503007 patients with untreated metastatic melanoma. [9] Based on those studies, ipilimumab was approved for metastatic melanoma in the United States and Europe in 2011. [4] However, when comparing to chemotherapy, tremelimumab failed to reach a higher survival advantage for metastatic melanoma. [10] Although the anti-CTLA-4 monoclonal antibodies indeed show a great effect on tumor immunity, patients often develop immune-related adverse events (irAEs) in tissues like skin, intestine, liver, and lung, which often have high microbial exposure. [11][12][13] Although irAEs can be fatal, corticosteroids and anti-inflammatory therapies can promote successful management of those toxicities in immune check point blockade. [4]

PD-1 AND PD-L1
Programmed death-1(PD-1, CD279) was first discovered by Tasuku Honjo and his colleagues. Similar to CTLA-4, PD-1 serves as a negative regulator for immune response, by mediating T cell exhaustion, T cell activation, T cell tolerance, and T cell resolution of inflammation. [4,14] To achieve this, PD-1 binds to its ligands, PD-L1(B7-H1, CD274) and PD-L2(B7-DC, CD273). When T cells receive repetitive stimulation from antigens in the setting of cancer and chronic infection, the level of PD-1 increases and T cells goes through epigenetic modification, which leads to a state called exhaustion. T cells in exhaustion state tend to express other inhibitory receptors, making them susceptible to inhibition by multiple checkpoint pathways. [4,15] PD-1 can be expressed on other cells as well, on NK cells and B cells, limiting their effector function [16][17][18], on macrophages, suppressing their function during diseases like sepsis, and on T regulatory cells and T follicular regulatory cells, modulating their induction and function. [4,19] The binding of PD-1 and PD-L1 or PD-L2 will cause the tyrosine phosphorylation of the PD-1 cytoplasmic area and recruitment of tyrosine phosphatase SHP-2, which leads a reduced downstream of T cell receptor stimulation, decreased phosphorylation of TCR signaling molecules, and lower production of cytokines. [20] Signaling of PD-1 can also trigger expression of proteins that inhibits T cell proliferation and production of cytokines and increase the expression of proapoptotic gene like Bcl2l11 (Bim), thus decreasing the overall survival of T cells. [21] Lastly, T regulatory cells from naïve or Th cells can be induced by PD-1 pathway, which can suppress the T cell effector functions. [4,19] Overall, PD-1 pathway can downregulate the activity of T cells in many ways to inhibit the effector function of T cells.
Several PD-1 monoclonal antibodies are approved to show great effect on immunotherapy, leading to FDA approvement. In 2014, nivolumab and pembrolizumab are approved for advances refractory melanoma and in 2015, nivolumab are approved for advanced refractory squamous NSCLC and renal cell carcinoma. [4] Pembrolizumab is an anti-PD-1 antibody that is being used to evaluate patients with renal cell carcinoma(RCC) in a phase II trial KEYNOTE-427 (NCT02853344). [22] Patients who received treatment with first-line pembrolizumab achieved an ORR of 38% and a disease control rate of 69% with a median duration of 12 months. [22,23] Atezolizumab, an anti-PD-L1 antibody, was used as a first-line single-agent for metastatic RCC in a phase I study (NCT01375842) and evaluated in a phase II IMmotion 150 trial (NCT01984242), including atezolizumab plus bevacizumab, atezolizumab alone or sunitinib. [22][23][24] Atezolizumab achieved an ORR OF 25% compared with sunitinib of 29%. [22]A randomized compartment of PD-1 and CTLA-4 blockade in melanoma shows the better effect of pembrolizumab over ipilimumab, with a higher response rate, lower rate of irAEs (19.9% versus 13.3% and 10.1%). [25] More therapeutic trials indicate the benefits and tumor immunity of PD-1/PD-L1 blockade.

26] LAG-3 is expressed on CD4+ and CD8+ Tcells, NK cells, NKT cells, and dendritic cells.[27] Its expression can be upregulated by interleukin (IL)-2 and IL-12 on activated T cells, where it mainly functions as a receptor that delivers inhibitory signals. LAG-3 can be also found on regulatory T-cells and helps to increase the suppressive ability of them.[28] LAG-3 downregulates the proliferation and function of T-cells and is associated with the exhaustion of T-cells.
It is worthy to note that PD-1 and LAG-3 are often co-expressed in tumor microenvironment. [29] LAG-3 is the homolog of CD4 that binds to MHC class II molecules, but with a higher affinity than CD4. [30] Except the canonical ligand MHC-II, other ligands have been discovered such as galectin-3, LSECtin, a-synuclein, and liver secreted protein fibrinogen-like protein 1 (FGL1). Among these ligands, FGL-1 was recently demonstrated as the major ligand of LAG-3 that mediates T cell suppression. [31] FGL1 is normally released into the blood plasma by the liver in low levels but by cancer in high levels. Though the physiological functions of FGL1 in hepatocyte regeneration and metabolism are not well understood, blockade the interaction of FGL1-LAG-3 potentiates antitumor immunity in a receptor-ligand inter-dependent manner. [31] The function of LAG-3 depends on the signaling through its cytoplasmic domain KIEELE motif, but the downstream signaling mechanism is still not clear. [28,32] Based on a study of murine LAG-3 Ig, Immutep (IMP321) was created and used to test in several clinical trials. In a study combined Immutep with taxane-based chemotherapy for women with breast cancer showed a result of increasing number and activation of APC and augmented number of NK cells and long-lived cytotoxicmemory CD8 T cell. [33] Additionally, the ORR was twice higher than the historical control group. There are at least ten clinical trials in development using LAG-3 monoclonal antibodies with combination with other agents like anti-PD-1 or anti-PD-L1. [28] The first E3S Web of Conferences 185, 03007 (2020) ICEEB 2020 http://doi.org/10.1051/e3sconf/202018503007 antagonistic mAb to LAG-3 was relatlimab. Combination of relalimab with nivolumab receive an ORR of 11.5% in patients with advanced melanoma in a phase I-II study. [28,34] Patients with expression of LAG-3 by TILs shows a higher response rate than patients without expression of LAG-3 on TILs. [28,34] Research have made several bispecific antibodies to target both LAG-3 and PD-1/PD-L1. FS118, a bispecific monoclonal antibody, is composed with anti-PD-L1 with its Fc region structured by anti-LAG-3. Studies have shown FS118 helps to enhance the activation of CD8+ T cells compared to anti-PD-L1 alone. [28,35]

SIRPΑ AND CD47
Signal-regulatory Protein alpha (SIRPα) is an inhibitory receptor that expresses on macrophages, dendritic cells, and neutrophils. [36] SIRPα binds to its ligand CD47, which is broadly expressed on many normal cells, but significantly expressed on cancer cell. [36] The pathway of SIRPα-CD47 is associated with a process known as phagocytosis. Phagocytosis is a process that cells like macrophages engulf many objects like cell debris, pathogen, red blood cells and most importantly tumor cells. The engulfment of those objects results in phagolysosome in which the object is digested and destroyed. It can also produce antigenic peptides that are presented on MHC class molecules, and then cause an initial adaptive immune response. The binding of SIRPα-CD47 is the inhibitory signal to the macrophage. Now, we understand the role of SIRPα-CD47 pathway in phagocytosis, but how does it operate under molecular level? The cytoplasmic domain of SIRPα has four tyrosine residues, which forms two ITIMs (immunoreceptor tyrosine-based inhibition motifs). [37] When they become phosphorylated, they recruit and activate inhibitory molecules, in particular tyrosine phosphatase SHP-1and SHP-2, which can dephosphorylate many substrates and decrease the downstream signaling pathways. [36,37] Although this inhibitory cascade finally restrain the function of nonmuscle myosin IIA [38] , the molecular intermediates via this pathway remain to be clarified. Overall, lack of binding of SIRPα-CD47 allows the activation of stimulatory receptors to trigger phagocytosis.
Several Abs and recombinant proteins, targeting CD47 or SIRPα, have been developed and are going through early clinical trials. [39] An anti-CD47 antibody, Hu5F9-G4, was shown to be efficiency, used either as single agent in patients with relapsed/refractory cancers in a phase I clinical trial [40], or in combination with the anti-CD20 monoclonal antibody rituximab for the treatment of relapsed or refractory non-Hodgkin lymphoma. [41] In addition, a pleiotropic anticancer agent in phase III clinical trials, RRx-001, has recently been recognized as a minimally toxic dual checkpoint inhibitor of CD47 and SIRPα that primes macrophages both in vitro and in vivo to attack cancer cells (Table  1). [42]

DISCUSSION
In recent years, many combinations of ICB were used for different types of cancers. One of the approaches for the future immunotherapy is to design better combinations of agents. However, ICB decreases the capability of the immune system to prevent autoimmunity and inflammation. New approaches should be to find receptors that are expressed specifically in the tumor sites, so the blockade can only happen in the tumor site. [11] Other researches that work on restricting tumor cells are developing strategies, including: (i) Vaccines targeting neoantigens [43][44][45]; (ii) better delivery of blocking agents to the tumor microenvironment; (iii) Chimeric antigen receptor T-cell therapy (CAR-T) [46]; (iv) bispecific antibodies [47][48][49]; and (v) Molecular shields restricting local activity of checkpoint inhibitors. [50] So, our scope of the future of immunotherapy is to suppress the activity of tumors while preventing autoimmunity.